Mutational profiles in HIV-1 protease correlated with phenotypic protease inhibitor resistance

ABSTRACT

The present invention is directed to the field of nucleic acid diagnostics and the identification of base variation in target nucleic acid sequences. More particularly, the present invention relates to the use of such genotypic characterization of a target population of HIV and the subsequent association, i.e., correlation, of this information to phenotypic interpretation in order to correlate virus mutational profiles with drug resistance. The invention also relates to methods of utilizing the mutational profiles of the invention in drug development, i.e., drug discovery, drug design, drug modification, and therapy, treatment design, clinical management and diagnostic analysis.

This application is the national stage of Application No.PCT/EP2003/050277, filed Jun. 30, 2003, which application claimspriority from U.S. Application Ser. No. 60/392,753, filed Jul. 1, 2002.

The present invention is directed to the field of nucleic aciddiagnostics and the identification of base variation in target nucleicacid sequences. The invention provides novel mutations or mutationalprofiles of HIV-1 protease gene correlated with a phenotype causingalterations in sensitivity to anti-HIV drugs. The present invention alsorelates to the use of genotypic characterization of a target populationof HIV and the subsequent association, i.e. correlation, of thisinformation to phenotypic interpretation in order to correlate virusmutational profiles with drug resistance. The invention further relatesto methods of utilizing the mutational profiles of the invention indatabases, drug development, i.e., drug design, and drug modification,therapy and treatment design and clinical management.

The development and standardization of plasma HIV-1 RNA quantificationassays has led to the use of viral load measurements as a key therapyresponse monitoring tool. The goal of antiretroviral therapy is toreduce plasma viremia to below the limit of detection on a long-termbasis. However, in a significant number of patients, maximal suppressionof virus replication is not achieved and for those in whom this goal isreached, a significant number experience viral load rebound. Viral loaddata provide no information on the cause of the failure.

Therapy failure may be due to a number of factors, includinginsufficient antiviral activity of the regimen, individual variations indrug metabolism and pharmacodynamics, difficulties in adhering to dosingregimen, requirements for treatment interruption due to toxicity, andviral drug resistance. Moreover, drug resistance may develop in apatient treated with sub-optimal antiretroviral therapy or a patient maybe infected with drug-resistant HIV-1. Although drug resistance may notbe the primary reason for therapy failure, in many cases any situationwhich permits viral replication in the presence of an inhibitor sets thestage for selection of resistant variants.

Viral drug resistance can be defined as any change in the virus thatimproves replication in the presence of an inhibitor. HIV-1 drugresistance was first described in 1989 and involved patients that hadbeen treated with zidovudine monotherapy (Larder, B. A., et al., Science243, 1731-1734 (1989)). Emergence of resistance is almost always beingobserved during the course of treatment of patients with singleantiretroviral drugs. Similarly, in vitro passage of viral culturesthrough several rounds of replication in the presence of antiretroviralcompounds leads to the selection of viruses whose replication cycle isno longer susceptible to the antiretroviral compounds used. Resistancedevelopment has also been observed with the introduction of dualnucleoside reverse transcriptase inhibitors (NRTI) combination therapyas well as during the administering of the more potent non-nucleosidereverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) andcombinations thereof. Individual antiretroviral agents differ in therate at which resistance develops: selection for resistant variants mayoccur within weeks of treatment or resistance may emerge after a longertreatment period.

Extensive genetic analysis of resistant viral isolates generated throughin vivo or in vitro selection has revealed that resistance is generallycaused by mutations at some specific site(s) of the viral genome. Themutational patterns that have been observed and reported for HIV-1 andthat are correlated with drug resistance are very diverse: someantiretroviral agents require only one single genetic change, whileothers require multiple mutations for resistance to appear. A summary ofmutations in the HIV genome correlated with drug resistance has beencompiled (See e.g. Schinazi, Int. Antiviral News. 6, 65 (2000)).Electronic listings with mutations are available at different weblocations such as hiv-web.lanl.gov/content/index, www.hivb.stanford.edu,and www.hivresistanceweb.com.

A genetic mutation is normally written in reference to the wild typevirus, i.e., K101N refers to replacement of a Lysine at codon 101 with aAsparagine (The Molecular Biology of the Cell, 1994, Garland Publishing,NY). However, the mutations of the invention do not depend on thewild-type example listed in order to be within the practice of theinvention. For example, the mutation 101N, refers to an Asparagine atthe 101 codon regardless of the whether there was a Lysine at 101 priorto mutation. Alternatively, it may be said that a particular amino acidoccurs at a given position, wherein “position” is equivalent to “codon”.Mutations can also be identified in nucleic acids such as RNA, DNA,mRNA.

The degree of susceptibility of a genetic variant to an antiretroviralcompound is expressed herein relative to the wild-type virus (HIVIIIB/LAI reference sequence) as found, for example, in GenBank, thesequence of which is hereby incorporated by reference (K03455, gi327742, M38432). An alteration in viral drug sensitivity is defined as achange in resistance or a change in susceptibility of a viral strain tosaid drug. Susceptibilities are generally expressed as ratios of EC₅₀ orEC₉₀ values (the EC₅₀ or EC₉₀ value being the drug concentration atwhich 50% or 90% respectively of the viral population is inhibited fromreplicating) of a viral strain under investigation compared to the wildtype strain. Hence, the susceptibility of a viral strain can beexpressed as a fold change in susceptibility, wherein the fold change isderived from the ratio of for instance the EC₅₀ values of a mutant viralstrain compared to the wild type. In particular, the susceptibility of aviral strain or population may also be expressed as resistance of aviral strain, wherein the result is indicated as a fold increase in EC₅₀as compared to wild type EC₅₀.

As antiretroviral drugs are administered for longer periods, mostly incombination with each other, and as new antiretrovirals are beingdeveloped and added to the present drugs, new resistance-correlatedgenetic variants are being identified. Of particular importance is thatthe combination of antiretroviral agents can influence resistancecharacteristics.

Once viral resistance has developed, salvage therapy options may beseverely restricted due to cross-resistance within each drug class. Thisis as important for initial treatment as for when a therapy change iscalled for in order to minimize the emergence of resistance and improvethe long-term prognosis of the patient. The choice of therapy regimenwill be supported by knowledge of the resistance profile of thecirculating virus population. Additionally, therapy combinations willhave a greater chance of being effective if they include agents thathave a demonstrated potential of suppressing a particular viruspopulation.

A number of applications describe the occurrence of mutations in HIV andtheir correlation to the development of drug resistance (WO 00/73511; WO02/33402; WO 02/22076; WO 00/78996). The instant invention adds to theart mutations in the protease gene and their correlation i.e.association to viral drug resistance.

DETAILED DESCRIPTION OF THE INVENTION

The knowledge that mutations at position 41 and 70 correlate with a foldchange in resistance can be applied in certain useful methods. Thepresent invention relates to methods for evaluating the effectiveness ofa protease inhibitor, based on the presence of at least one mutationselected from 41S, 41T, 41I, 41K, 41G and 70E, in HIV protease. Inparticular, the present invention relates to methods for evaluating theeffectiveness of a protease inhibitor, based on the presence of at leastone mutation selected from 41T, 41I, 41K, 41G and 70E, in HIV protease.The presence of at least one of said mutations correlates to a foldchange in susceptibility or resistance of an HIV viral strain towards atleast one protease drug. The effectiveness of a protease inhibitor inthe presence of at least one of said mutations may be determined usinge.g. enzymatic, phenotypic and genotypic methods. The correlationbetween the mutational profiles in HIV protease and drug usage may beuseful for clinical toxicological and forensic applications. A combinedapproach involving genotypic and phenotypic resistance testing tocorrelate mutations with resistance phenotypes may be used. More inparticular, the present invention provides a correlation between atleast one strain of HIV having at least one mutation in HIV proteaseselected from 41T and 70E and a fold change in resistance. In one aspectof the invention, the HIV protease mutations, 41T and 70E, are bothpresent in a viral strain.

The effectiveness of a protease inhibitor as an antiviral therapy for apatient infected with at least one HIV strain comprising mutant proteasemay be determined using a method comprising: (i) collecting a samplefrom an HIV-infected patient; (ii) determining whether the samplecomprises a HIV protease having at least one mutation selected from 41S,41T, 41I, 41K, 41G, and 70E; and (iii) correlating the presence of saidat least one mutation of step (ii) to a change in effectiveness of saidprotease inhibitor. In particular, the effectiveness of a proteaseinhibitor as an antiviral therapy for a patient infected with at leastone HIV strain comprising mutant protease-may be determined using amethod comprising: (i) collecting a sample from an HIV-infected patient;(ii) determining whether the sample comprises a HIV protease having atleast one mutation selected from 41T, 41I, 41K, 41G, and 70E; and (iii)correlating the presence of said at least one mutation of step (ii) to achange in effectiveness of said protease inhibitor.

In general a change in effectiveness can be expressed as a fold changein resistance. The fold change may be determined using a cellular assayincluding a cytopathogenic assay or the Antivirogram® (WO 97/27480).Alternatively, the fold change in susceptibility may be derived fromdatabase analysis such as the VirtualPhenotype™ (WO 01/79540). Adecrease in susceptibility vis-à-vis the wild type virus correlates toan increased viral drug resistance, and hence reduced effectiveness ofsaid drug. To determine the viral drug susceptibility the activity ofthe mutant enzyme may be compared to the activity of a wild type enzyme.In phenotyping assays pseudotyped viruses may be used. The mutationspresent in HIV protease may be determined at the nucleic acid or aminoacid level using sequencing or hybridization techniques. A report may begenerated that shows the region of the patient virus that has beensequenced, including at least one mutation selected from 41S, 41T, 41I,41K, 41G and 70E, in particular, including at least one mutationselected from 41T, 41I, 41K, 41G and 70E. The report may includeantiretroviral drugs, drug(s) for which a known resistance-associatedmutation has been identified and/or to what extent the observedmutations selected from at least 41S, 41T, 41I, 41K, 41G and 70E areindicative of resistance to said drugs. In particular, the report mayinclude drug(s) for which a known resistance-associated mutation hasbeen identified and/or to what extent the observed mutations selectedfrom at least 41T, 41I, 41K, 41G and 70E are indicative of resistance tosaid drugs. HIV may be present in combinations of several strains. Thismay result in the presence of multiple mutations at a particular aminoacid, including partial mutations. Partial mutations include thecombination of the wild amino acid and a mutant amino acid at aparticular position. Examples thereof include partial mutations atposition 41 in HIV protease, including 41R/S, 41S/R, 41R/K, 41G/R, inparticular 41R/K, 41G/R. The sample to be evaluated can be a bodilyfluid including blood, serum, plasma, saliva, urine, or a tissueincluding gut tissues.

The fact that particular data correlate, indicates that a causalrelationship exits between the data. Hence, a particular result rendersa particular conclusion more likely than other conclusions.

A drug effective against mutant HIV protease may be identified by amethod, comprising: (i) providing a nucleic acid comprising HIV proteasecomprising at least one mutation chosen from 41S, 41T, 41I, 41K, 41G and70E; (ii) determining a phenotypic response to said drug for said HIVrecombinant virus; and (iii) identifying a drug effective against mutantHIV based on the phenotypic response of step (ii). In particular, a drugeffective against mutant HIV protease may be identified by a method,comprising: (i) providing a nucleic acid comprising HIV proteasecomprising at least one mutation chosen from 41T, 41I, 41K, 41G and 70E;(ii) determining a phenotypic response to said drug for said HIVrecombinant virus; and (iii) identifying a drug effective against mutantHIV based on the phenotypic response of step (ii). The nucleic acidcomprising HIV of step (i) may be recombined into a proviral nucleicacid deleted for said sequence to generate a recombinant HIV virus.

Identifying a drug is defined as making a selection of drugs clinicallyavailable based on the effectiveness of said drug. In addition to theselection of clinically available drugs, identifying also relates to theselection of clinical drug candidates. The phenotypic response may bedetermined using cellular assays such as the Antivirogram®. An effectivedrug against mutant HIV comprising at least one mutation in proteaseselected from 41T and 70E, is defined as a drug having a phenotypicresponse expressed, as e.g. a fold change in susceptibility lower than adefined cut-off that may be determined for a drug.

An other useful method for identifying a drug effective against mutantHIV protease comprising:

-   -   (i) providing a HIV protease comprising at least one mutation        chosen from 41S, 41T, 41I, 41K, 41G and 70E;    -   (ii) determining the activity of said drug on said HIV protease;    -   (iii) determining the activity of said drug on wild type HIV        protease;    -   (iv) determining the ratio of the activity determined in        step (iii) over the activity determined in step (ii);    -   (v) identifying an effective drug against mutant HIV based on        the ratio of step (iv).

In particular, a useful method for identifying a drug effective againstmutant HIV protease comprising:

-   -   (i) providing a HIV protease comprising at least one mutation        chosen from 41T, 41I, 41K, 41G and 70E;    -   (ii) determining the activity of said drug on said HIV protease;    -   (iii) determining the activity of said drug on wild type HIV        protease;    -   (iv) determining the ratio of the activity determined in        step (iii) over the activity determined in step (ii);    -   (v) identifying an effective drug against mutant HIV based on        the ratio of step (iv).

A ratio lower than a defined cut-off value that can be specific for saiddrug is indicative that the drug is effective against mutant HIV (WO02/33402).

The activity of said drug on said HIV protease, having at least onemutation selected from 41S, 41T, 41I, 41K, 41G and 70E, in particular41T, 41I, 41K, 41G and 70E, can be determined in an enzymatic assay,wherein the mutant protease, is compared to the wild type enzyme by itsenzymatic characteristics (e.g. Maximal velocity (V_(max)),Michaelis-Menten constant (K_(m)), catalytic constant (k_(cat))). Anactivity means any output generated by the assay including fluorescence,fluorescence polarization, luminiscence, absorbance, radioactivity,resonance energy transfer mechanisms, magnetism. The use of fluorescentsubstrates to measure the HIV protease activity was described by e.g.Matayoshi et al. [Science 1990, 247, 954], Tyagi et al. [Anal. Biochem.1992, 200(1), 143], Toth et al. [Int. J. Pept. Protein Res. 1990, 36(6),544] and Wang et al. [Tetrahedron 1990, 31(45), 6493] and in severalpatent applications [see e.g. WO99/67417; EP428000, EP518557]. Asuitable substrate for the enzymatic determination isR-E(EDANS)-S-Q-N-Y-P-I-V-Q-K(DABCYL)-R—OH (Science, 1989, 247, 954-958).Alternatively HPLC based methods may be used to determine the activity.

The response of a mutant HIV protease having at least one mutationselected from 41S, 41T, 41I, 41K, 41G and 70E, in particular 41T, 41I,41K, 41G and 70E, may be expressed as viral fitness (WO 00/78994). Thisviral fitness can be defined as the ability of a viral strain toreplicate in the presence or absence of a component, such as a proteaseinhibitor. This viral fitness is dependent on a combination of factorsincluding viral factors which include mutations occurring in viralproteins, such as the mutations described herein, host factors whichinclude immune responses, differential expression of membrane proteinsand selective pressures which include the presence of antiviral agentssuch as protease inhibitors.

Interestingly, protease inhibitors that can be used in the presentmethods include Nelfinavir, Saquinavir, Indinavir, Amprenavir,Tipranavir, Lopinavir, Ritonavir, Palinavir, Atazanavir, Mozenavir,Fosamprenavir, compound 1 (Carbamic acid,[(1S,2R)-3-[[(4-aminophenyl)sulfonyl](2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)propyl]-,(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl ester, compound 1), andcompound 2, which has been described as a HIV protease inhibitor inWO02/083657 and which can be prepared according to the proceduresdescribed therein. Compound 2 has the following chemical structure:

In an embodiment, the protease inhibitor is selected from Indinavir,Saquinavir, Lopinavir, Nelfinavir, compound 1, and compound 2. Inparticular, the protease inhibitor is selected from Indinavir,Saquinavir, Lopinavir and compound 1.

Conveniently, the methods of the present invention are performed usingsamples of an HIV-infected patient that has been treated with at least aprotease inhibitor. More in particular, the patient contains mutantviruses bearing at least one additional mutation at position in the HIVprotease selected from 10, 30, 33, 46, 47, 50, 54, 63, 71, 74, 77, 82,84, 88 or 90. Even more in particular, the mutant viruses are resistanttowards the therapy the patient is taken.

A vector comprising an HIV sequence having at least one mutation in theHIV protease gene chosen from 41S, 41T, 41I, 41K, 41G and 70E may beuseful for the phenotypic analysis. In particular, a vector comprisingan HIV sequence having at least one mutation in the HIV protease genechosen from 41T, 41I, 41K, 41G and 70E may be useful for the phenotypicanalysis. The present knowledge about the correlation between a foldchange in susceptibility and the presence of at least one mutationselected from 41S, 41T, 41I, 41K, 41G and 70E in HIV protease can beused to prepare an isolated and purified HIV protease sequence having atleast one mutation selected from 41S, 41T, 41I, 41K, 41G and 70E. Inparticular, the knowledge about the correlation between a fold change insusceptibility and the presence of at least one mutation selected from41T, 41I, 41K, 41G and 70E in HIV protease can be used to prepare anisolated and purified HIV protease sequence having at least one mutationselected from 41T, 41I, 41K, 41G and 70E.

The knowledge of the mutations of the present invention offers thepossibility to develop probes and primers directed to said mutations. Anisolated and purified oligonucleotide comprising a HIV protease sequenceof 5 to 100 bases comprising at least one mutation chosen from 41S, 41T,41I, 41K, 41G and 70E, may be useful for in vitro diagnosis of viraldrug resistance. In particular, an isolated and purified oligonucleotidecomprising a HIV protease sequence of 5 to 100 bases comprising at leastone mutation chosen from 41T, 41I, 41K, 41G and 70E, may be useful forin vitro diagnosis of viral drug resistance. Suitable oligonucleotidesfor nucleic acid amplifying technologies contain 5 to 35 nucleic acidbases. Suitably such oligonucleotides contain 15 to 30 nucleic acidbases. An oligonucleotide may contain the mutant base at the 3′ end soas to enable the detection of the mutant using PCR. Oligonucleotides mayalso be used as probes including molecular beacons (Tyagi, NatureBiotechnol 1998, 16(1) 49-53), and TaqMan probes.

A computer system comprising at least one database correlating thepresence of at least one mutation in a human immunodeficiency virusprotease and fold change in susceptibility of at least one strain of HIVto a protease inhibitor, comprising at least one record corresponding toa correlation between at least one mutation selected from 41S, 41T, 41I,41K, 41G and 70E, in particular 41T, 41I, 41K, 41G and 70E, andtreatment with at least a protease inhibitor can be used for evaluatingresistance towards therapy.

A neural network that predicts the development of therapeutic agentresistance or sensitivity against at least one viral strain comprisingat least one mutation selected from 41S, 41T, 41I, 41K, 41G and 70E canbe used (WO 01/95230). In particular, a neural network that predicts thedevelopment of therapeutic agent resistance or sensitivity against atleast one viral strain comprising at least one mutation selected from41T, 41I, 41K, 41G and 70E can be used (WO 01/95230).

Genotyping Methodologies

Resistance of HIV to antiretroviral drugs may be determined at thegenotypic level by identifying mutations in the HIV-1 genome and byinferring the resistance of HIV-1 to antiretroviral drugs throughsearching for mutational patterns known to correlate with resistance.Assays for detection of mutations in HIV-1 may be based on polymerasechain reaction (PCR) amplification of viral genomic sequences. Theseamplified sequences are then analyzed using either hybridization orsequencing techniques. Hybridization-based assays includeprimer-specific PCR, which makes use of synthetic oligonucleotidesdesigned to allow selective priming of DNA synthesis. See Larder, B. A.,et al., AIDS 5, 137-144 (1991); Richman, D. D., et al., J. Infect. Dis.164, 1075-1081 (1991); Gingeras, T. R., et al., J. Infect. Dis. 164,1066-1074 (1991). Only when primer sequences match the target sequence(wild-type or mutant) at the 3′ end, is amplification of targetsequences possible and DNA fragments are produced. Knowledge of theprimer sequences allows one to infer the sequence of the viral isolateunder investigation, but only for the region covered by the primersequences. Other hybridization-based assays include differentialhybridization (Eastman, P. S., et al., J. Acq. Imm. Def. Syndr. HumanRetrovirol. 9, 264-273 (1995); Holodniy, M., et al., J. Virol. 69,3510-3516 (1995); Eastman, P. S., et al., J. Clin. Micro. 33, 2777-2780(1995).); Line Probe Assay (LiPA® HIV-11 RT, Innogenetics) (Stuyver, L.,et al., Antimicrob. Agents Chemotherap. 41, 284-291 (1997)); and biochiptechnology such as GENECHIP® technology (Affymetrix) (D'Aquila, R. T.Clin. Diagnost. Virol. 3, 299-316 (1995); Fodor, S. P. A. et al., Nature364, 555-556 (1993); Fodor, S. P. A. Nature 227, 393-395 (1997). Thesequence may also be determined using mass spectroscopic technologies.DNA sequencing assays provide information on all nucleotides of thesequenced region. Sequencing results may be reported as amino acidchanges at positions in the protease gene and the reverse transcriptasegene compared to the wild-type reference sequence. The changes includedin the genotyping report may be limited to mutations at positions knownto manifest drug resistance-associated polymorphisms. Polymorphisms atpositions not associated with drug resistance may be omitted.

Phenotyping Methodologies

Phenotyping assays measure the ability of a replicating virus to grow inthe presence of compounds compared to a wild-type reference virus suchas e.g. HIV-1/LAI, HIV-1/NL4.3, HIV-1/HXB2 or e.g. HIV-2/ROD.Alternatively, phenotyping assays are performed with pseudotyped virusesnot able to replicate (WO 02/38792). Consequently, these assays directlymeasure the degree of viral susceptibility to specific inhibitors. Inthis case, one measures the effect of all mutational interactions, theeffects of genetic changes as yet unknown or not previously identified,the effect of the background genotype, etc., on the phenotype. Somephenotypic assays are discussed below.

Cytopathic Effect Assay (CPE Assay)

Determination of the antiviral activity of a compound was done asdescribed in Pauwels R. et al. (J Virol Methods 1988; 20(4):309-21).Various concentrations of the test compounds were brought into each wellof a flat-bottom microtiter plate. Subsequently, HIV and MT4 cells wereadded to a final concentration of 200-250 50% cell culture infectiousdoses (CCID₅₀)/well and 30,000 cells/well, respectively. After 5 days ofincubation (37° C., 5% CO₂), the cytopathic effect of the replicatingvirus was determined by the tetrazolium colorimetric MTT method. Thedose protecting 50% of the cells from virus cytopathic effect wasdefined as the EC₅₀, while the dose achieving 90% protection was definedas the EC₉₀.

Reporter Gene Assay

The reporter gene assay used MT4-LTR-EGFP cells. Upon infection byHIV-1, the expression of the viral tat product increases transcriptionfrom the HIV-1 LTR promoter, leading to high-level expression of thereporter gene product. The assay procedure was similar to the CPE assay,except for the end reading of the assay, which was performed on day 3 bymeasuring the relative fluorescence of treated cultures and comparingthis with the relative fluorescence of untreated cultures. The EC₅₀ orthe EC₉₀ of a compound was defined as the concentration that inhibitedthe relative fluorescence by 50% or 90% respectively.

Antiviral Assay with PBMC Cultures

The purification and activation of PBMCs as well as the antiviral assayswere carried out as described (CDER. Guidance for Industry Points toConsider in the Preclinical Development of Antiviral Drugs. 1990). Theassay measured the extent that a drug inhibits HIV p24 antigenproduction by peripheral blood mononuclear cells (PBMC) cultures acutelyinfected with a viral strain. The susceptibility determination usesphytohaemaglutinine (PHA)-stimulated PBMCs from normal donors. In the invitro infection experiments 1000 CCID₅₀ per million PHA-stimulated PBMCswas used. Cultures were split ½ every 3 to 4 days and compound was addedtogether with the addition of new medium.

The p24 antigen production was measured using a commercial kit,according to the manufacturer protocol (NEN), at the moment that the p24production of untreated infected cultures is maximal; i.e. between 7 and11 days after infection.

The % p24 production was calculated by means of following equation:

${\%\mspace{14mu}{p24}} = {100 \times \frac{\lbrack{p24}\rbrack_{Sample} - \lbrack{p24}\rbrack_{Mock\_ Control}}{\lbrack{p24}\rbrack_{HIV\_ Control} - \lbrack{p24}\rbrack_{Mock\_ Control}}}$where [p24]_(Sample) is the p24 concentration in an infected treatedculture, [p24]_(HIV) _(—) _(Control) is the p24 concentration in aninfected untreated culture and [p24]_(Mock) _(—) _(Control) is the p24concentration in a mock-infected culture. The dose achieving 50% p24production according to the above formula was defined as the EC₅₀, whilethe dose achieving 10% p24 production according to the above formula wasdefined as the EC₉₀.Antiviral Assay with Monocytes/Macrophages

The assay measured the extent that a drug inhibits HIV p24 antigenproduction by primary monocytes/macrophages acutely infected withHIV-1/BaL (300 CCID₅₀/ml). The susceptibility determination usedmonocytes/macrophages isolated from PBMCs from normal donors by plasticadherence. Every 5 days cultures were fed with complete mediumcontaining the appropriate compound concentrations. The p24 antigenproduction was measured at day 14 after virus challenge and EC₅₀ andEC₉₀ values were calculated.

Recombinant Virus Assays

A recombinant virus assay (RVA) starts with the amplification of viraltarget sequences by means of PCR. The amplicons are incorporated into aproviral laboratory clone deleted for the sequences, present in theamplicon. This generates a stock of recombinant viruses. The viruses aretested for their ability to grow in the presence of differentconcentrations of drugs. Results are obtained by calculating EC₅₀ valuesfor each inhibitor and by reporting the results as EC₅₀ values,expressed in μM concentrations, or by computing the ratio of the EC₅₀values found for the recombinant virus to the EC₅₀ values found for awild type susceptible laboratory virus tested in parallel. In the lattercase, resistance is expressed as “fold-resistance” (fold change insusceptibility, FC) compared to a wild-type susceptible HIV-1 strain.

The use of reporter gene systems for susceptibility testing allows theimplementation of laboratory automation and standardization (Pauwels, etal., J. Virol. Methods 20, 309-321 (1988); Paulous, S., et al.,International Workshop on HIV Drug Resistance, Treatment Strategies andEradication, St. Petersburg, Fla., USA. Abstr. 46 (1997); and Deeks, S.G., et al., 2nd International Workshop on HIV Drug Resistance andTreatment Strategies, Lake Maggiore, Italy. Abstr. 53 (1998)).

The Antivirogram® assay (Virco) (WO 97/27480) is based on homologousrecombination of patient derived HIV-1 gag/PR/RT sequences into aproviral HIV-1 clone correspondingly deleted for the gag/PR/RTsequences. A similar assay (Phenosense® ViroLogic, WO 97/27319) is basedon enzymatic ligation of patient-derived PR/RT sequences into acorrespondingly deleted proviral vector carrying an indicator gene,luciferase, inserted in the deleted HIV-1 envelope gene. Another assaywas developed by Bioalliance (Phenoscript, WO 02/38792). The developmentof high throughput phenotyping and genotyping assays has allowed theestablishment of a database containing the phenotypic resistance dataand the genotypic sequences of over 30,000 clinical isolates.

Experimental Part

EXAMPLE 1 The Identification of Mutational Patterns in HIV-1 Proteaseand the Correlated Phenotypic Resistance

Plasma samples from HIV-1-infected individuals from routine clinicalpractice were obtained and shipped to the laboratory on dry ice andstored at −70° C. until analysis. Viral RNA was extracted from 200 μLpatient plasma using the QIAAMP® Viral RNA Extraction Kit (Qiagen,Hilden, Germany), according to the manufacturers instructions. cDNAencompassing part of the pol gene was produced using Expand™ reversetranscriptase (Boehringer Mannheim). A 2.2 kb fragment encoding theprotease and RT regions were amplified from patient-derived viral RNA bynested polymerase chain reaction (PCR) using PCR primers and conditionsas described. (Hertogs K., et al., Antimicrob. Agents Chemother. 42:269-276 (1998), WO 01/81624). This genetic material was used inphenotyping and genotyping experiments.

Phenotypic analysis was performed using the recombinant virus assay(Antivirogram®)(WO 97/27480). MT-4 cells (Harada S., et al, Science 229:563-566 (1985).) were co-transfected with pol gene PCR fragments and theprotease-RT deleted HIV-1 molecular clone, pGEM3ΔPRT. This resulted inviable recombinant viruses containing protease/RT from the donor PCRfragment. After homologous recombination of amplicons into a PR-RTdeleted proviral clone, the resulting recombinant viruses wereharvested, titrated and used for in vitro susceptibility testing toantiretroviral drugs. The results of this analysis were expressed asfold change in susceptibility, reflecting the fold change in mean EC₅₀(μM) of a particular drug when tested with patient-derived recombinantvirus isolates, relative to the mean EC₅₀ (μM) of the same drug obtainedwhen tested with a reference wild-type virus isolate (IIIB/LAI).

Genotyping was performed by an automated population-based full-sequenceanalysis, through a dideoxynucleotide-based approach, using the BigDye™terminator kit (Applied Biosystems, Inc.) and resolved on an ABI 377 DNAsequencer.

The genotypes are reported as amino acid changes at positions along theprotease gene compared to the wild-type (HXB2) reference sequence.Analysis by VirtualPhenotype™ interpretational software (WO 01/79540)allowed detection of mutational patterns in the database containing thegenetic sequences of the clinical isolates and linkage with thecorresponding resistance profiles of the same isolates.

EXAMPLE 2 Susceptibility Analysis of HIV-1 Variants Constructed bySite-Directed Mutagenesis

Mutations in the protease or RT coding region were created bysite-directed mutagenesis, using the QuikChange® Site-DirectedMutagenesis Kit (STRATAGENE®), of a wild-type HXB2-D EcoRl-Pstlrestriction enzyme fragment, encompassing the HIV-1 pol gene and clonedinto pGEM3 (Promega). All mutant clones were verified by DNA sequenceanalysis. PCR fragments were prepared from the mutated clones and thealtered protease coding regions were transferred into HIV-1 HXB2-D byhomologous recombination as described above. The susceptibility of theserecombinant viruses to drugs was determined by the MT-4 cell CPEprotection assay.

EXAMPLE 3 In Vitro Selection of Resistant Strains

MT4-LTR-EGFP cells were infected at a multiplicity of infection (MOI) of0.01 to 0.001 CCID₅₀/cell in the presence of inhibitor. The startingconcentration of the inhibitor was two to three times the EC₅₀, asuboptimal concentration. The cultures were sub-cultivated and scoredmicroscopically on virus-induced fluorescence and cytopathicity every3-4 days. The cultures were sub-cultivated in the presence of the samecompound concentration until signs of virus replication were observed.The escaping virus was further cultivated in the presence of the sameinhibitor concentration in order to enrich the population in resistantvariants. If full virus breakthrough was observed the supernatant wascollected and stored (new virus strain). Afterwards, the same virus waschallenged with a higher compound concentration in order to selectvariants able to grow in the presence of as high as possible inhibitorconcentrations. From the new viruses, a virus stock was grown in theabsence of inhibitor.

In vitro drug selection experiments starting from wild-type HIV-1 underpressure of compound 1, compound 2, and Nelfinavir (NFV) have beenperformed. Tables 1, 2, 3, 4, and 5 show the genotypic and phenotypiccharacterization of the selected strains.

TABLE 1 Characterization of the strains isolated from HIV-1/LAI in thepresence of compound 1 In vitro selection Experimental conditionsStarting strain HIV/ HIV-1/LAI HIV-1/LAI HIV-1/LAI LAI Compound —Compound 1 Compound 1 Compound 1 Concentration (nM) — 30 100 100 Days —45 97 188 Protease Genotype Mutations — R41T R41T R41T K70E K70EPhenotype In vitro susceptibility to PIs N, median EC50 (nM), median FCCompound 1 N 37 7 6 3 EC50 3.2 7.7 26 44 FC 1 2 8 10 Indinavir N 16 3 32 EC50 28 33 98 140 FC 1 1 4 5 Ritonavir N 16 3 3 2 EC50 31 32 21 46 FC1 1 1 1 Nelfinavir N 11 3 4 2 EC50 30 32 18 37 FC 1 1 1 1 Saquinavir N46 2 6 3 EC50 7.8 30 35 150 FC 1 4 4 20 Amprenavir N 67 3 6 3 EC50 36 3829 39 FC 1 1 1 1 Lopinavir N 11 3 5 3 EC50 7.9 27 32 47 FC 1 3 4 6

TABLE 2 Characterization of the strains isolated from HIV-1/LAI in thepresence of compound 1 In vitro selection Experimental conditionsStarting strain HIV-1/ HIV-1/ HIV-1/ HIV-1/ HIV-1/ LAI LAI LAI LAI LAICompound — Comp 1 Comp 1 Comp 1 Comp 1 Concentration (nM) — 30 100 100200 Days — 70 139 195 328 Protease Genotype Mutations — S37S/N S37N S37NR41R/K R41S R41S K70E K70E K70E K70E Phenotype In vitro susceptibilityto PIs N, median EC₅₀ (nM), median FC Compound 1 N 5 1 1 1 1 EC50 2.66.5 2.5 4.7 0.4 FC 1 3 1 2 0.2 IDV N 4 1 1 2 1 EC50 12 18 6.3 9.1 5.5 FC1 2 1 1 0.5 RTV N 3 1 1 2 1 EC50 33 47 22 14 31 FC 1 1 1 0.4 1 NFV N 4 11 2 1 EC50 38 39 9.7 9.5 1.9 FC 1 1 0.3 0.3 0.1 SQV N 3 1 1 1 1 EC50 5.66.0 0.7 0.9 4.0 FC 1 1 0.1 0.2 1 APV N 5 1 1 2 1 EC50 20 56 24 14 15 FC1 3 1 1 1 LPV N 5 1 1 2 1 EC50 4.6 17 2.8 3.9 1.1 FC 1 4 1 1 0.2

TABLE 3 Characterization of the strains isolated from HIV-1/LAI in thepresence of compound 2 In vitro selection Experimental conditionsStarting strain HIV-1/LAI HIV-1/LAI HIV-1/LAI HIV-1/LAI Compound —Compound 2 Compound 2 Compound 2 Concentration — 100 100 100 (nM) Days —116 200 264 Protease Genotype Mutations — G16G/H G16E R41I R41I R41IPhenotype In vitro susceptibility to PIs N, median EC₅₀ (nM), median FCCom- N 2 2 1 pound 2 EC50 12 6.9 61 FC 1 1 5 IDV N 4 1 1 EC50 12 19 47FC 1 2 4 RTV N 3 2 1 EC50 33 22 23 FC 1 1 1 NFV N 4 2 1 EC50 38 16 14 FC1 0 0 SQV N 3 1 EC50 5.6 45 FC 1 8 APV N 5 2 1 EC50 20 14 8.4 FC 1 1 0LPV N 5 2 1 EC50 4.6 <0.9 18 FC 1 0 4

TABLE 4 Characterization of the strains isolated from HIV-1/LAI in thepresence of compound 1 In vitro selection Experimental conditionsStarting strain HIV-1/ HIV-1 HIV-1 HIV-1 HIV-1 LAI Compound — — Comp 1Comp 1 Comp 1 Concentration (nM) — — 20 40 40 Days — — 94 161 175Protease Genotype Mutations — — R41G/R R41G R41G V82V/I V82I V82IPhenotype In vitro susceptibility to PIs N, median EC₅₀ (nM), median FCCompound 1 N 5 1 1 1 1 EC50 2.6 3.4 1.1 2.6 1.9 FC 1 1 0 1 1 IDV N 4 1 11 1 EC50 12 2.4 3.1 2.1 3.9 FC 1 0 0 0 0 RTV N 3 1 1 1 1 EC50 33 22 4.26.3 1.7 FC 1 1 0 0 0 NFV N 4 1 1 1 1 EC50 38 31 5.7 11 16 FC 1 1 0 0 0SQV N 3 1 1 1 1 EC50 5.6 8.9 0.9 1.0 0.8 FC 1 2 0 0 0 APV N 5 1 1 1 1EC50 20 26 7.3 6.6 9.4 FC 1 1 0 0 0 LPV N 5 1 1 1 1 EC50 4.6 6.8 2.0 1.81.0 FC 1 1 0 0 0

TABLE 5 Characterization of the strains isolated from HIV-1/LAI in thepresence of nelfinavir (NFV) In vitro selection Experimental conditionsStarting strain HIV-1/ HIV-1/ HIV-1/ HIV-1/ HIV-1/ LAI LAI LAI LAI LAICompound — NFV NFV NFV NFV Concentration (nM) — 1000 3000 9000 9000 Days— 35 69 111 140 Protease Genotype Mutations — L10F L10F L10F D30N D30ND30N D30N R41R/K R41R/K K45I/K K45I/K M46I M46I M46I M46I V77I V77I V77II84V/I I84V I85V/I I85V/I N88D/ N88D N88D N Phenotype In vitrosusceptibility to PIs N, median EC₅₀ (nM), median FC IDV N 4 1 1 1 EC5012 7.9 100 28 FC 1 1 8 2 RTV N 3 1 1 1 1 EC50 33 19 27 86 170 FC 1 1 1 35 NFV N 4 1 1 1 EC50 38 330 7200 6800 FC 1 9 200 200 SQV N 3 1 1 1 1EC50 5.6 1.8 2.5 15 34 FC 1 0 0 3 6 APV N 5 1 1 1 1 EC50 20 28 59 95 190FC 1 1 3 5 10 LPV N 5 1 1 1 1 EC50 4.6 7.7 24 39 56 FC 1 2 5 8 10 The invitro antiviral activity of compound 1, compound 2, Nelfinavir, andcurrent PIs against the selected strains was evaluated in acutelyinfected MT4 cells. Median EC₅₀ values together with the number ofdeterminations (N), and the fold change in EC₅₀ as compared to wild type(FC) are reported.

1. A method for evaluating the effectiveness of a protease inhibitor asan antiviral therapy for a patient infected with at least one mutant HIVstrain comprising: (i) collecting a sample from an HIV-infected patient;(ii) extracting the nucleic acid from said patient sample; (iii)determining the amino acid sequence encoded by said nucleic acid; (iv)determining whether said amino acid sequence comprises at least onemutation selected from R41S, R41T, R41I, R41G and K70E in the proteaseregion; (v) measuring the effectiveness of said protease inhibitoragainst said mutant HIV strain; (vi) correlating the presence of said atleast one mutation of step (iv) to a change in the effectiveness of saidprotease inhibitor against said mutant HIV strain relative to theeffectiveness of said protease inhibitor against a wild type HIV strain(HIV IIIB/LAI reference sequence).
 2. A method for evaluating theeffectiveness of a protease inhibitor as an antiviral therapy for apatient infected with at least one mutant HIV strain comprising: (i)collecting a sample from an HIV-infected patient; (ii) extracting thenucleic acid from said patient sample; (iii) determining the amino acidsequence encoded by said nucleic acid; (iv) determining whether saidamino acid sequence comprises at least one mutation selected from R41T,R41I, R41G and K70E in the protease region; (v) measuring theeffectiveness of said protease inhibitor against said mutant HIV strain;(vi) correlating the presence of said at least one mutation of step (iv)to a change in the effectiveness of said protease inhibitor for saidpatient relative to the effectiveness of said protease inhibitor for apatient infected with a wild type HIV strain (HIV IIIB/LAI referencesequence).
 3. A method for evaluating a change in the susceptibility ofa HIV strain to a protease inhibitor comprising the steps of: (i)collecting a sample from an HIV-infected patient; (ii) extracting thenucleic acid from said patient sample; (iii) determining the amino acidsequence encoded by said nucleic acid; (iv) determining whether saidamino acid sequence comprises at least one mutation selected from R41S,R41T, R41G and K70E in the protease region; (v) measuring thesusceptibility of said HIV strain from said patient to said proteaseinhibitor; (vi) correlating the presence of said at least one mutationof step (iv) to a change in susceptibility of the patient HIV strain tosaid protease inhibitor relative to the susceptibility of a wild typeHIV strain (HIV IIIB/LAI reference sequence) to said protease inhibitor.4. A method for evaluating a change in the susceptibility of a HIVstrain to a protease inhibitor comprising the steps of: (i) collecting asample from an HIV-infected patient; (ii) extracting the nucleic acidfrom said patient sample; (iii) determining the amino acid sequenceencoded by said nucleic acid; (iv) determining whether said amino acidsequence comprises at least one mutation selected from R41T, R41G andK70E in the protease region; (v) measuring the susceptibility of saidHIV strain from said patient to said protease inhibitor; (vi)correlating the presence of said at least one mutation of step (iv) to achange in susceptibility of the patient HIV strain to said proteaseinhibitor relative to the susceptibility of a wild type HIV strain (HIVIIIB/LAI reference sequence) to said protease inhibitor.